Gene Cloning, Prokaryotic Expression and Purification of ABC Transporter in Haemonchus contortus
ZHAO Xue-Liang1,2, WANG Shu-Yi1, SUN Ke1, Huhe Bateer1, WANG Wen-Long1,*, LIU Chun-Xia3,*
1 College of Veterinary Medicine, Inner Mongolia Agricultural University/Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, Ministry of Agriculture and Rural Affairs, Hohhot 010018, China; 2 College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China; 3 College of Life Science, Inner Mongolia Agricultural University, Hohhot 010018, China
Abstract：Haemonchus contortus is one of the most important parasites of ruminants such as Ovis aries and Capra hircus, and can bring huge economic losses to the breeding industry. In preliminary transcriptome study of present research group, the resistant gene ATP-binding cassette (ABC) transporter gene (GenBank No. MT478136) was identified from the resistant strain of Haemonchus contortus. The purpose of this study was to explore the bioinformatics character of the ABC transporter gene, to establish a system of soluble expression and purification for ABC transporter, and to lay the foundation for further exploration of ABC transporter-involved drug resistance mechanism. The online software was used to predict the protein structure and antigen epitope. The results showed that the molecular formula of ABC was C928H1502N270O265S7, which was a hydrophilic protein without transmembrane region and signal peptide. The secondary structure was mainly extended chains. The prediction of antigenic epitopes indicated that ABC transporter had many antigenic epitopes with high antigenicity. The ABC transporter gene was amplified by reverse transcription PCR (RT-PCR), and the recombinant plasmid pET30a(+)-ABC was constructed by restriction digestion. The plasmid was transformed into Escherichia coli BL21 to optimize inducing conditions. It was found that 1 mg/mL recombinant ABC transporter could be expressed with 0.6 mmol/L isopropyl β-D-thiogalactoside (IPTG) at 37 ℃ for 7 h. In this study, recombinant ABC transporter was successfully expressed and purified in Haemonchus contortus, which could provide a reference for further mechanism study on potential drug resistance and immunogenicity.
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